ABSTRACT
Influenza virus utilizes a unique mechanism for initiating the transcription of viral mRNA. The viral transcriptase ribonucleoprotein complex hydrolyzes host cell transcripts containing the cap 1 structure (m7GpppG(2'-OMe)-) to generate a capped primer for viral mRNA transcription. Basic aspects of this viral endonuclease reaction are elucidated in this study through the use of synthetic, radiolabeled RNA substrates and substrate analogs containing the cap 1 structure. Unlike most ribonucleases, this viral endonuclease is shown to catalyze the hydrolysis of the scissile phosphodiester, resulting in 5'-phosphate- and 3'-hydroxyl-containing fragments. Nevertheless, the 2'-OH adjacent to the released ribosyl 3'-OH is shown to be important for catalysis. In addition, while the endonuclease steady-state turnover rate is measured to be 2 h(-1), phosphodiester bond hydrolysis is not rate-limiting. The direct generation of a free 3'-OH and the subsequent slow release of this product are consistent with the viral need for efficient use of the capped primer in subsequent reactions of the influenza transcriptase complex.
Subject(s)
Endoribonucleases/metabolism , Oligoribonucleotides/chemical synthesis , Oligoribonucleotides/metabolism , Orthomyxoviridae/enzymology , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , RNA/metabolism , Autoradiography , Base Sequence , DNA Primers , Kinetics , Molecular Sequence Data , Oligoribonucleotides/chemistry , Phosphorus Radioisotopes , RNA/chemical synthesis , RNA Caps/metabolism , Substrate Specificity , Transcription, GeneticABSTRACT
Many different formulation techniques are available for designing controlled-release dosage forms. Five different erosion-controlled or diffusion-controlled delivery systems were evaluated to select the 1 most suitable for Sinemet CR. The system ultimately selected, containing carbidopa-levodopa 50-200 mg, is a monolithic matrix tablet designed to have both of its active components released by surface dissolution and erosion. This system was found to be the most effective following extensive in vitro testing, pharmacokinetic studies, and clinical trials. Sinemet CR releases both carbidopa and levodopa by a 1st-order release rate. Controlled-release dosage forms of levodopa with slower in vitro release rates have lower plasma levels.
